Antioxidant effects of carnitine supplementation on 14-3-3 protein isoforms in the aged rat hippocampus detected using fully automated two-dimensional chip gel electrophoresis.

We here described the antioxidant effects of carnitine supplementation on 14-3-3 protein isoforms in the aged rat hippocampus detected using the fully automated two-dimensional chip gel electrophoresis system (Auto2D). This system was easy and convenient to use, and the resolution obtained was more sensitive and higher than that of conventional two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). We separated and identified five isoforms of the 14-3-3 protein (beta/alpha, gamma, epsilon, zeta/delta, and eta) using the Auto2D system. We then examined the antioxidant effects of carnitine supplementation on the protein profiles of the cytosolic fraction in the aged rat hippocampus, demonstrating that carnitine supplementation suppressed the oxidation of methionine residues in these isoforms. Since methionine residues are easily oxidized to methionine sulfoxide, the convenient and high-resolution 2-D PAGE system can be available to analyze methionine oxidation avoiding artifactual oxidation. We showed here that the Auto2D system was a very useful tool for studying antioxidant effects through proteomic analysis of protein oxidation.

Phosphorus recovery by mesoporous structure material from wastewater.

This study was designed to investigate the fundamental aspects of a possible removal of phosphorous from wastewater by using the mesoporous structure materials for wastewater reuse. The zirconium sulphate with mesoporous structure as a new type of ion exchangers was synthesised by hydrothermal reaction. From the results of X-ray diffraction and transmission electron microscope, it was discovered that the synthesised material had hexagonal mesoporous structure with a pore size of approximately 40-50A. Experimental results showed that the zirconium sulphate with mesoporous structure had very high sorption capacity for the phosphorus. The novel ion exchange occurred between PO4(3-) and SO4(2-), OH-. The amount of phosphate ions exchanged into the solid was as great as 3.4 mmol/g-ZS. Furthermore, it is possible to obtain a higher removal efficiency than other ion exchange media and adsorbents.

Feeding unsaponifiable compounds from rice bran oil does not alter hepatic mRNA abundance for cholesterol metabolism-related proteins in hypercholesterolemic rats.

The hypocholesterolemic effect of rice bran oil (RBO) is defined in human and animal experiments which indicate the presence of active component(s) in the unsaponifiable fraction, but the detailed mechanism is not known yet. Exogenously hypercholesterolemic (ExHC) rats were fed for 2 weeks on a 0.5% cholesterol diet supplemented with 10% each of RBO, RBO-simulated oil (RBOSO) in its fatty acid composition, or RBOSO plus 0.25% unsaponifiable compounds (UC) from RBO. Rats fed RBO or the UC resulted in lowing serum and liver cholesterol concentration and preventing reduction of high density lipoproteinic-cholesterol. Dietary RBO or the UC led to an elevation of fecal neutral sterol excretion, but no significant change in fecal bile acid excretion or in hepatic abundance of mRNAs for 3-hydroxy-3-methylglutaryl-CoA reductase, cholesterol-7alpha-hydroxylase, and low density lipoprotein receptor. Besides, serum and liver alpha-tocopherol concentrations were lowered in RBO or the UC-fed rats. These results show that the UC in RBO leads to a decreased serum cholesterol concentration by interrupting the absorption of intestinal hydrophobic compounds rather than by modifying cholesterol metabolism in the liver.

Cancer chemopreventive agents, labdane diterpenoids from the stem bark of Thuja standishii (Gord.) Carr.

Seven labdane-type diterpenoids from the stem bark of Thuja standishii (Gord.) Carr. (Cupressaceae) and their analogues showed strong inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). Among these compounds, 15,16-bisnor-13-oxolabda-8(17), 11E-dien-19-oic acid was revealed to have the strongest inhibitory effect on the EBV-EA activation, being stronger than that of beta-carotene which has been intensively studied in cancer prevention using animal models. 15,16-bisnor-13-Oxolabda-8(17), 11E-dien-19-oic acid was also found to exhibit the excellent anti-tumor promoting activity in two-stage mouse skin carcinogenesis test using 7,12-dimethylbenz[a]anthracene and TPA.

Comparison of methotrexate resistance conferred by a mutated dihydrofolate reductase (DHFR) cDNA in two different retroviral vectors.

We previously reported the protection of hematopoietic cells from methotrexate (MTX) toxicity using an N2-based double copy vector containing serine 31 (S31)-mutated dihydrofolate reductase (DHFR) (DC/SV6S31). To examine whether the use of SFG-based dicistronic vectors will lead to improvement in gene transfer over the DC/SV6 vector, we compared the protection provided by MTX to NIH3T3 cells and hematopoietic progenitor cells infected with these retroviral constructs containing the S31 variant DHFR cDNA. In NIH3T3 cells, the 50% effective dose values of MTX conferred by the SFG vector were 8-fold higher than those obtained with the DC/SV6 vector. DHFR mRNA levels were 22-fold and 38-fold higher than that seen for the DC/SV6 vector according to Northern blot and real-time polymerase chain reaction analysis, respectively. However, DHFR protein expression and DHFR enzyme activity were only 1.5-fold and 2-fold higher in the SFG vector, respectively, indicating that the mRNA from the SFG vector is translated less efficiently than the mRNA generated from the DC/SV6 vector. Furthermore, the degree of MTX protection conferred by each vector in both mouse and human hematopoietic cells was the same. These results indicate that the in vitro transduction efficiency and transgene expression of human DHFR in hematopoietic progenitor cells is equally conferred by both vectors.

Cbfa1 is a positive regulatory factor in chondrocyte maturation.

Cbfa1 is a transcription factor that belongs to the runt domain gene family. Cbfa1-deficient mice showed a complete lack of bone formation due to the maturational arrest of osteoblasts, demonstrating that Cbfa1 is an essential factor for osteoblast differentiation. Further, chondrocyte maturation was severely disturbed in Cbfa1-deficient mice. In this study, we examined the possibility that Cbfa1 is also involved in the regulation of chondrocyte differentiation. mRNAs for both Cbfa1 isotypes, type I Cbfa1 (Pebp2alphaA/Cbfa1) and type II Cbfa1 (Osf2/Cbfa1 or til-1), which are different in N-terminal domain, were expressed in terminal hypertrophic chondrocytes as well as osteoblasts. In addition, mRNA for type I Cbfa1 was expressed in other hypertrophic chondrocytes and prehypertrophic chondropcytes. In a chondrogenic cell line, ATDC5, the expression of type I Cbfa1 was elevated prior to differentiation to the hypertrophic phenotype, which is characterized by type X collagen expression. Treatment with antisense oligonucleotides for type I Cbfa1 severely reduced type X collagen expression in ATDC5 cells. Retrovirally forced expression of either type I or type II Cbfa1 in chick immature chondrocytes induced type X collagen and MMP13 expression, alkaline phosphatase activity, and extensive cartilage-matrix mineralization. These results indicate that Cbfa1 is an important regulatory factor in chondrocyte maturation.

Decreased food intake and body weight in pancreatic polypeptide-overexpressing mice.

BACKGROUND & AIMS: Pancreatic polypeptide (PP) is a 36-amino acid hormone produced by F cells within the pancreatic islets and the exocrine pancreas. The definitive function of PP in mammalian physiology remains to be determined. This study examined the effects of chronic overexpression of PP through the development of PP transgenic mice. METHODS: PP transgenic mice were created by using mouse PP complementary DNA under the control of the cytomegalovirus immediate early enhancer-chicken beta-actin hybrid promoter (pCAGGS expression vector). RESULTS: A unique line of transgenic mice was created that overexpresses PP in the pancreatic islets with low levels of expression in other tissues including the brain. Plasma PP concentrations were more than 20 times higher than those of control littermates. However, PP overproduction led to postnatal lethality in half of the pups because of markedly decreased milk intake. The remaining PP transgenic mice gained less weight with specifically reduced food intake and fat mass compared with controls, a result that was more evident in male than in female mice. The transgenic mice exhibited a reduced rate of gastric emptying of a solid meal but had normal oxygen consumption and fasting leptin levels. Immunoneutralization with anti-PP antiserum reversed the phenotypic changes of transgenic animals. CONCLUSIONS: PP could be involved in feeding and body weight regulation partly through regulation of gastric emptying.

A comparison of blood pressure changes in phacoemulsification surgery with topical and retrobulbar block local anesthesia: part II.

Although we recently compared blood pressure (BP) changes during cataract surgery between groups that received topical and retrobulbar block anesthesia, a study has not been conducted in which patients were matched for age and sex. To draw more meaningful conclusions, we conducted an age- and sex-matched study in which the daily, pre- and postoperative blood pressures of 1,398 cataract patients were compared. All surgeries were performed using the same method of phacoemulsification and aspiration with intraocular lens implantation under local anesthesia. The postoperative BPs of the retrobulbar injection group decreased significantly more than the topical application group. Even when the patients were hypertensive, the postoperative BPs decreased. Following retrobulbar block anesthesia, the BP decreased postoperatively to a greater extent than after topical anesthesia.

Differential inhibitory mechanisms in VPL versus intralaminar nociceptive neurons of the cat: I. Effects of periaqueductal gray stimulation.

Nociceptive thalamic units receiving afferent input from the greater splanchnic nerve (SPL) were recorded from the nucleus ventralis posterolatealis (VPL) and intralaminar nuclei in urethane-chloralose anesthetized cats. The effects of stimulating the periaqueductal gray (PAG), or the nucleus raphe dorsalis (NRD) on responses of nociceptive thalamic units were investigated. Forty-eight nociceptive specific (NS) and 20 wide dynamic range (WDR) units with SPL input were found in the shell region of the caudal VPL. Following electrical stimulation of either the ventral PAG or the NRD, responses to SPL input were inhibited in all NS and WDR units tested. Responses of these units to electrical stimulation of spinothalamic tract fibers in the ventrolateral funiculus (VLF) were also inhibited following the PAG/NRD stimulation. These results suggest that PAG/NRD stimulation-produced inhibition of both NS and WDR units may be partially mediated by an ascending antinociceptive mechanism. Intralaminar nociceptive units with SPL input were found in the nuclei centralis lateralis (CL), paracentralis (Pc), and parafascicularis (Pf). The effects of conditioning electrical stimulation of either the ventral PAG or the NRD on responses of intralaminar nociceptive units were studied. Of 113 intralaminar nociceptive units studied, 68 units were unaffected, 23 units were excited and 22 units were inhibited following the conditioning stimulation. In the units in which responses to SPL stimulation were inhibited, responses elicited by electrical stimulation of the mesencephalic reticular formation (MRF) were also inhibited. These data suggest that although there is an ascending inhibitory pathway from PAG/NRD to intralaminar nuclei, this system is far less potent compared with the ascending inhibitory system acting upon the VPL.

Retinoic acid treatment induces type X collagen gene expression in cultured chick chondrocytes.

The vitamin A derivative retinoic acid (RA) is widely thought to be involved in cartilage development, but its precise roles and mechanisms of action in this complex process remain unclear. We have tested the hypothesis that RA is involved in chondrocyte maturation during endochondral ossification and, in particular, is an inducer of maturation-associated traits such as type X collagen and alkaline phosphatase. Immature chondrocytes isolated from the caudal region of Day 19 chick embryo sterna were seeded in secondary monolayer cultures and treated either with a high dose (100 nM) or with physiological doses (10-35 nM) of RA for up to 3 days. We found that after an initial lag of about 24 h, physiological doses of RA indeed induced type X collagen gene expression in the immature cells. This induction was not accompanied by obvious changes in expression of the type II collagen and large aggregating proteoglycan core protein genes. As revealed by immunocytochemistry, 30-35% of the cells in cultures treated with RA for 3 days were engaged in type X collagen production. Interestingly, these cells were relatively similar in size to chondrocytes in which no type X collagen was detected, suggesting that chondrocytes can initiate type X collagen production independent of cell hypertrophy. RA treatment also led to increased alkaline phosphatase activity occurring as early as 24 h after the start of treatment. The data in this study indicate that RA may have a role in endochondral ossification as an inducer/promoter of maturation-associated traits during chondrocyte maturation.

The growth inhibition of Streptococcus mutans by 5'-nucleotidase inhibitors from Areca catechu L.

New 5'-nucleotidase inhibitors named NF-86I, NF-86II were recently isolated from the seeds of Areca catechu L. NF-86I and NF86II showed inhibitory effects on the growth of Streptococcus mutans MT8148(c) and Streptococcus mutans MT6715(g), respectively. In addition, these inhibitors could inhibit insoluble glucan formation from sucrose. NF-86I and NF-86II were found to be polyphenolic substances. Some polyphenols such as tannic acid bind non-specifically to proteins (tannic activity). The 5'-nucleotidase inhibitors that we isolated did not show any such activity. However, the growth inhibitory activity and the inhibitory effect on water-insoluble glucan production were equal to tannic acid. It is therefore considered that these inhibitors bind specifically to the bacterial cell surface. Our findings suggest that the 5'-nucleotidase inhibitors NF-86I and NF-86II may be useful anti-plaque preventing agents.

Effects of acupuncture on immune response in mice.

The effect of acupuncture points stimulation on the induction of plaque-forming cells (PFC) in spleen cells of BALB/c mice was investigated in vivo and in vitro tests. In in vivo experiment, mice were immunized with 2 x 10(8) sheep red blood cells (SRBC) and the PFC was markedly increased by daily (once a day for 4 days) acupuncture stimulation. The enhancement of PFC by acupuncture was completely blocked by preadministration of procain, hexamethonium, naloxone, propranolol, but not by phentolamine. The enhancement of PFC by acupuncture was also observed in spleen cells of non-immunoized mice when spleen cells of the acupunctured mice were cultured with SRB in vitro. The enhancement of PFC in spleen was observed after stimulation with acupuncture, and a similar effect was also found in bone marrow cells of normal mice, but not in thymic cells. The spleen cells of mice given acupuncture showed no enhancement of PFC after treatment with anti-Thy 1.2 antibody and complement. Furthermore, these helper T cells were found to be not restricted by the H-2 gene complex. These data demonstrate that the helper T cells induced by acupuncture lack the H-2 restriction, and thus suggest that they may be drived from the bone marrow, but not from the thymus. It is therefore concluded that the helper T cells derived from the bone marrow were activated via the sympathetic nervous system stimulated by acupuncture.

Stimulation by concanavalin A of cartilage-matrix proteoglycan synthesis in chondrocyte cultures.

The effect of concanavalin A on proteoglycan synthesis by rabbit costal and articular chondrocytes was examined. Chondrocytes were seeded at low density and grown to confluency in medium supplemented with 10% fetal bovine serum, and then the serum concentration was reduced to 0.3%. At the low serum concentration, chondrocytes adopted a fibroblastic morphology. Addition of concanavalin A to the culture medium induced a morphologic alteration of the fibroblastic cells to spherical chondrocytes and increased by 3- to 4-fold incorporation of [35S]sulfate and [3H]glucosamine into large chondroitin sulfate proteoglycan that was characteristically found in cartilage. The stimulation of incorporation of labeled precursors reflected real increases in proteoglycan synthesis, as chemical analyses showed a 4-fold increase in the accumulation of macromolecules containing hexuronic acid in concanavalin A-maintained cultures. Furthermore, the effect of concanavalin A on [35S]sulfate incorporation into proteoglycans was greater than that of various growth factors or hormones. However, concanavalin A had smaller effects on [35S]sulfate incorporation into small proteoglycans and [3H]glucosamine incorporation into hyaluronic acid and chondroitinase AC-resistant glycosaminoglycans. Since other lectins tested, such as wheat germ agglutinin, lentil lectin, and phytohemagglutinin, had little effect on [35S]sulfate incorporation into proteoglycans, the concanavalin A action on chondrocytes seems specific. Although concanavalin A decreased [3H]thymidine incorporation in chondrocytes, the stimulation of proteoglycan synthesis could be observed in chondrocytes exposed to the inhibitor of DNA synthesis, cytosine arabinoside. These results indicate that concanavalin A is a potent modulator of proteoglycan synthesis by chondrocytes.

Vasodilatory active principles of Sophora flavescens root.

Flavanones vexibinol and kurarinone were isolated from an ethyl acetate extract of Sphora flavescens roots and assayed using isolated rabbit and rat thoracic aorta helical strips. These compounds inhibited KCl and norepinephrine maximum contractions in a concentration-dependent manner in the rat but only KCl contractions in the rabbit. Vexibinol and kurarinone weakly inhibited histamine- and serotonin-induced contractions in the rabbit. The results suggest that these flavanones may inhibit Ca2+ influx through a voltage-dependent Ca2+ channel.

Hypertrophy and calcification of rabbit permanent chondrocytes in pelleted cultures: synthesis of alkaline phosphatase and 1,25-dihydroxycholecalciferol receptor.

Cartilage calcification at specific sites is a key event that leads to skeletal development and growth. To obtain insights into the control of cartilage calcification, we examined whether cells distributed in permanent cartilage regions might have the ability to express the calcification-related phenotype in a permissive environment. Chondrocytes were isolated from the permanent and growth plate cartilages of 4-week-old rabbit ribs. They were seeded as a pelleted mass in a centrifuge tube and cultured in Eagle's minimum essential medium supplemented with 10% fetal bovine serum. These cells proliferated for several generations, and then synthesized large amounts of proteoglycans, yielding a cartilage-like tissue in 16 days. Cultures from the permanent and growth plate cartilages showed similar time courses for increases in DNA synthesis and proteoglycan production that reached similar maximal levels. Thereafter, they initiated the syntheses of alkaline phosphatase and 1,25-dihydroxycholecalciferol receptor and induced matrix calcification without additional phosphate. The increases in alkaline phosphatase, 1,25-dihydroxycholecalciferol receptor, and calcium contents in cultures from the permanent cartilage were consistently delayed for 4-7 days relative to the growth plate-derived cells, but caught up by Day 28. The maximal levels of alkaline phosphatase and 1,25-dihydroxycholecalciferol receptor in the cultures from the permanent cartilage were 40- to 100-fold higher than that of the in vivo permanent cartilage. These results provide evidence that permanent cartilage cells in postnatal young rabbit ribs have the capacity to express alkaline phosphatase and 1,25-dihydroxycholecalciferol receptor and induce calcification in a permissive environment, although they never express these calcification-related phenotypes in vivo.

Vascular dilatory action of the Chinese crude drug. II. Effects of scoparone on calcium mobilization.

Further experiments were conducted to examine the effect of scoparone (6,7-dimethoxycoumarin), a coumarin derivative found in the Chinese crude drug "Capillaris Flos," on calcium mobilization. Scoparone does not affect Ca2+ influx through the voltage-dependent channel due to membrane depolarization. Its inhibitory action may be dependent not only on the inhibition of Ca2+ release from sarcoplasmic reticulum but also on the inhibition of extracellular Ca2+ influx through the receptor-operated channel.

Stimulation of cartilage-matrix proteoglycan synthesis by morphologically transformed chondrocytes grown in the presence of fibroblast growth factor and transforming growth factor-beta.

The effects of transforming growth factor-beta (TGF-beta) on the synthesis of cartilage-matrix proteoglycan by cultured rabbit chondrocytes were examined. Rabbit chondrocytes were seeded at low density and exposed to a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium supplemented with 0.5% fetal bovine serum, 1% bovine serum albumin, 50 micrograms/ml ascorbic acid, and 2 x 10(-7) M hydrocortisone (Medium A). Various combinations of TGF-beta, insulin-like growth factor-I (IGF-I), and fibroblast growth factor (FGF) were also added to Medium A, and the chondrocytes were grown to confluency. Chondrocytes grown with TGF-beta or FGF alone became flat or fibroblastic, those grown with FGF and TGF-beta became very elongated and formed distinct foci, and those grown with FGF and IGF-I showed the spherical configuration characteristic of overtly differentiated chondrocytes. Nevertheless, the incorporation of 3H with glucosamine into the large, chondroitin sulfate proteoglycan synthesized by cultures with FGF and TGF-beta was similar to that in cells grown with FGF and IGF-I and five times that in cells cultured with FGF alone. The increases in incorporation of 3H reflected real increases in proteoglycan synthesis, because chemical analyses showed an increase in the accumulation of macromolecules containing uronic acid in cultures with FGF and TGF-beta or with FGF and IGF-I. However, FGF in combination with either TGF-beta or IGF-I had little effect on the incorporation of 3H into small proteoglycans or hyaluronic acid. These results indicate that chondrocytes morphologically transformed with TGF-beta and FGF fully express the differentiated proteoglycan phenotype rather than the transformed glycosaminoglycan phenotype.

Terminal differentiation and calcification in rabbit chondrocyte cultures grown in centrifuge tubes: regulation by transforming growth factor beta and serum factors.

Rabbit chondrocyte cultures on plastic dishes are capable of depositing a cartilaginous matrix, although the matrix does not calcify unless high levels of phosphate are added to the medium. In the present study, we cultivated a pelleted mass of rabbit growth-plate chondrocytes in the presence of Eagle's minimum essential medium supplemented with 10% fetal bovine serum and 50 micrograms of ascorbic acid per ml in a plastic centrifuge tube. These cells proliferated for several generations and then reorganized into a cartilage-like tissue that calcified without additional phosphate. The deposition of minerals was observed only after synthesis of a short-chain collagen and alkaline phosphatase. Serum factors were required for the increases in alkaline phosphatase and calcium contents. 5-Bromo-2'-deoxyuridine abolished the increases in uronic acid, alkaline phosphatase, and calcium contents. Transforming growth factor beta, at very low concentrations, suppressed the expression of the mineralization-related phenotype by chondrocytes. These results suggest that cartilage-matrix calcification can be controlled by growth factor(s) and that chondrocytes induce the mineralization of extracellular matrix when terminal differentiation is permitted in the absence of an artificial substrate.